Effects of C-methylated carnitine analogs on rates of mitochondrial fatty acid oxidation.

Carnitine analogs containing one and/or two methyl substituents on the alpha-, beta-, or gamma-carbon were evaluated in isolated rat liver mitochondria for their effects on fatty acid oxidation. Their abilities to either support, in the absence of carnitine, or inhibit, in the presence of carnitine, carnitine-dependent fatty acid oxidation were determined by the conversion of radiolabeled [1-14C]palmitic acid to acid-soluble radiolabeled products. None of the methylcarnitine analogs were observed to be significant inhibitors of palmitate oxidation at concentrations (1.0 mM) up to ten times that for L-carnitine. The two diastereomers of D,L-4-methylcarnitine, however, were able to support palmitate oxidation in the absence of carnitine, and rates were roughly 40% of that obtained with equimolar (0.1 mM) L-carnitine.

Energy dependent insulin binding, internalization and degradation in isolated cardiac myocytes from normal and diabetic rats.

Insulin binding to isolated cardiac myocytes from normal and streptozotocin-induced diabetic rats was investigated. We found that at high affinity sites, the maximum numbers of insulin binding sites per cell are 33 000 and 22 000 for normal and diabetic myocytes, respectively with no discernible difference in receptor affinity. However, since the yield of myocytes from the diabetic heart was only 1/3 of the normal heart, it is suspected that the insulin function in the diabetic heart may be significantly lower than that in the normal heart. Chloroquine was found to markedly decrease insulin degradation with concomitant increase in net insulin uptake by isolated myocytes. This suggests that insulin degradation may take place within lysosomes after insulin is internalized. To determine whether internalization of insulin in myocytes is an energy dependent process, insulin binding and subsequent degradation were assessed in cells depleted of ATP by treatment with various metabolic inhibitors (2,4-dinitrophenol, NaF and iodoacetic acid). Depletion of the cellular ATP level resulted in a decrease in both insulin uptake and degradation. In diabetic myocytes, the general relationship between cellular ATP level and insulin uptake and degradation was similar to that found in normal myocytes. However, in diabetic myocytes, the cellular ATP level and insulin uptake were lower, but insulin degradation was greater than in normal myocytes. Insulin uptake by normal and ATP depleted cells at 4 degrees C (16 h) was lower than at 37 degrees C (1 h), while the ATP level was almost the same at both temperatures. This suggests that the internalization of insulin is a temperature as well as an ATP dependent process.

Direct effects of streptokinase on the isolated rat myocardium.

The utilization of streptokinase as a thrombolytic agent is prevalent in the management of patients following a myocardial infarction. The present investigation was designed to assess the direct effects of streptokinase infusion on the normal isolated rat heart and on the globally ischemic isolated rat heart. The effects of streptokinase on the mechanical function of the hearts were monitored, as well as the effects on the membrane integrity of the cells, as indicated by enzyme release. The normal hearts were infused with either buffer or streptokinase. Both the normal and streptokinase-treated normal hearts exhibited a decrease in function during the course of each experiment. Under these conditions, there were no significant differences in the values for mechanical function in either group. The release of lactate dehydrogenase (LDH) and creatine kinase (CK) into the coronary effluents was not significantly increased during the infusion of streptokinase. There was a significant increase in the release of nucleosides during the streptokinase infusion period compared with the buffer infusion period. The infusion of either buffer or streptokinase was compared in the preischemic and postischemic rat heart. The values obtained during preischemic infusion with buffer or streptokinase were comparable to the values obtained in the normal and streptokinase-treated normal hearts. There were no significant differences in the values for mechanical function measured during buffer infusion following ischemia compared with those measured during streptokinase infusion following ischemia. In the postischemic hearts, there was a sixfold increase in the release of nucleosides during buffer infusion, whereas only a twofold increase was observed in the postischemic hearts during streptokinase infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of beta 1-adrenergic receptors from adult, rat cardiac myocytes.

The beta-receptors were isolated from rat cardiac myocytes and characterized. Isolated myocytes were prepared from adult rat hearts and characterized for viability. Membrane proteins were solubilized from myocytes with 1% Triton X-102. The solubilized membrane proteins were fractionated by DEAE-Sephacel ion exchange column chromatography. Two major protein peaks were obtained. The second protein peak sample was found to contain beta-receptors to which 125I-15-(4'-azido-3'-iodobenzyl)-carazolol (125I-ABC) was specifically bound. This sample was labeled covalently with 125I-ABC by UV irradiation. The radiolabeled sample was applied to a Sepharose CL-6B gel column. Two radiolabeled protein peaks, one with a molecular weight of approximately 570,000 and the other with a molecular weight of approximately 95,000 were found. When the 570,000-dalton complex was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, it was dissociated into a component with a molecular weight of 66,000. The 95,000-dalton complex was dissociated into a 58,000-dalton component upon SDS-PAGE under reducing conditions. An excess amount of isoproterenol and propranolol decreased photolabeling of the beta-receptors with 125I-ABC by 60% and 40%, respectively.